Many methods for peptide synthesis are described in the literature (for examples, see U.S. Pat. No. 6,015,881; Mergler et al. (1988) Tetrahedron Letters 29:4005-4008; Mergler et al. (1988) Tetrahedron Letters 29:4009-4012; Kamber et al. (eds), Peptides, Chemistry and Biology, ESCOM, Leiden (1992) 525-526; Riniker et al. (1993) Tetrahedron Letters 49:9307-9320; Lloyd-Williams et al. (1993) Tetrahedron Letters 49:11065-11133; and Andersson et al. (2000) Biopolymers 55:227-250; Bray, Brian L., Nature Reviews 2:587-593 (2003). The various methods of synthesis are distinguished by the physical state of the phase in which the synthesis takes place, namely liquid phase or solid phase.
Liquid phase methods (often referred to as solution phase methods) of synthesis carry out all reactions in a homogeneous phase. Successive amino acids are coupled in solution until the desired peptide material is formed. During synthesis, successive intermediate peptides are purified by precipitation and/or washes.
In solid phase peptide synthesis (SPPS), a first amino acid or peptide group is bound to an insoluble support, such as a resin. Successive amino acids or peptide groups are added to the first amino acid or peptide group until the peptide material of interest is formed. The product of solid phase synthesis is thus a peptide bound to an insoluble support. Peptides synthesized via SPPS techniques are then cleaved from the resin, and the cleaved peptide is isolated.
In addition to the liquid phase and SPPS techniques described above, a hybrid approach can be utilized. Hybrid synthesis is typically utilized to manufacture complex sequences. For example, in one representative hybrid scheme, complex sequences can be manufactured through the solid phase synthesis of protected peptide intermediates, which are subsequently assembled either by solution phase or SPPS methods to produce a longer peptide product. Thus, for example, as a step in the synthesis, an intermediate compound is produced that includes each of the amino acid residues located in its desired sequence in the peptide chain with various of these residues having side chain protecting groups. The protected peptide intermediates are then coupled in solution to form a longer peptide. See, for example, WO 99/48513.
Peptides and amino acids from which peptides are synthesized tend to have reactive side groups as well as reactive terminal ends. When synthesizing a peptide, it is important that the amino group on one peptide react with the carboxyl group on another peptide. Undesired reactions at side groups or at the wrong terminal end of a reactant produces undesirable by-products, sometimes in significant quantities. These can seriously impair yield or even ruin the product being synthesized from a practical perspective. To minimize side reactions, it is conventional practice to appropriately mask reactive side groups and terminal ends of reactants to help ensure that the desired reaction occurs.
For example, a typical solid phase synthesis scheme involves attaching a first amino acid or peptide group to a support resin via the carboxyl moiety of the peptide or amino acid. This leaves the amino group of the resin-bound material available to couple with additional amino acids or peptide material. Thus, the carboxyl moiety of the additional amino acid or peptide material desirably reacts with the free amino group of the resin-bound material. To avoid side reactions involving the amine group of the additional amino acid or peptide, such amine group is masked with a protecting group during the coupling reaction. Two well-known amine protecting groups are the BOC group and the FMOC group. Many others have also been described in the literature. After coupling, the protecting group on the N-terminus of the resin-bound peptide can be removed, allowing additional amino acids or peptide material to be added to the growing chain in a similar fashion. In the meantime, reactive side chain groups of the amino acid and peptide reactants, including the resin-bound peptide material as well as the additional material to be added to the growing chain, typically remain masked with side chain protecting groups throughout synthesis. These same concepts (without a support resin for the initial amino acid) can be applied to liquid phase synthesis techniques.
The step of removing protecting groups from a peptide is commonly referred to as deprotection. When all of the protecting groups (including terminal protecting groups and side chain protecting groups) are removed, this is referred to as global deprotection. In some cases, only the N-terminal protecting group is removed. The reagents utilized in N-terminal deprotection typically leave the side chain protecting groups intact. In one exemplary N-terminal deprotection scheme, the removal of the N-terminal protecting group (for example, an Fmoc group) is typically accomplished by treatment with a reagent that includes 20-50% (on a weight basis) piperidine in a solvent, such as N-methylpyrrolidone (NMP) or dimethylformamide (DMF). After removal of the Fmoc protecting group, several washes are typically performed to remove residual piperidine and Fmoc by-products (such as dibenzofulvene and its piperidine adduct). Conventional synthesis techniques have stressed the importance of removing residual piperidine, to reduce unwanted reactions, such as premature removal of Fmoc protecting groups on subsequent amino acids to be added to the peptide chain. In other words, it has been recognized that the Fmoc group should remain on amino acids until the particular amino acid has been incorporated into a growing peptide material and is ready to be activated for coupling to a subsequent amino acid.
Several tests have been developed to determine when the removal of Fmoc by-products and residual piperidine from a reaction solution is complete. The most common of these tests is the chloranil test, which utilizes a saturated solution of chloranil and toluene in acetone. Color is used to determine the presence of secondary amine (thus indicating that Fmoc by-products and/or residual piperidine are still present). Other tests involve monitoring the pH of the reaction solution to determine the presence of piperidine (for example, using pH paper). Still other tests involve including a dye with the deprotection reagent, and monitoring the color of the reaction solution visually to determine when the dye (and thus deprotection reagent) has been removed from the reaction solution. Once the Fmoc protecting group is removed, an additional activated amino acid residue can be added to the peptide fragment, and the cycle repeated for subsequent amino acid residues until the desired peptide is completed.
For large-scale production of peptides, issues relating to reagent consumption, as well as cycle and processing time, can greatly impact the feasibility of the peptide synthesis scheme. Thus, there is a continuing need for peptide synthesis processes capable of producing peptide materials of commercial interest in large batch quantities. Deprotection of amino acid residues at the N-terminus to allow coupling of an additional amino acid, for example, by treatment with a base, is one aspect of the synthesis in which improvement is needed. Conventional methodologies may utilize reagents at levels that are higher than desirable and involve additional processing steps that are unnecessary.